LOW TOXICITY OF NITRIC-OXIDE AGAINST ENDOTHELIAL-CELLS UNDER PHYSIOLOGICAL OXYGEN PARTIAL PRESSURES

Cultured rat liver endothelial cells were incubated with 1 and 2 mM sp ermineNONOate at different O-2 concentrations in the incubation atmosp here. ropylammonio)butyl]-amino}diazen-1-ium-1,2-diolate (spermineNONO ate), at 2 mM, was highly cytotoxic at 21 % and 95 % O-2 (as measured by lactate dehydrogenase release); more than 80 % of the cells were da maged after 6 h of incubation. Cytotoxicity induced by 2 mM spermineNO NOate was significantly decreased at 10 %, 5 % and 0 % O-2; cell death was 54 %, 36 % and 25 % respectively after 6 h of incubation. In cont rast, 1 mM spermineNONOate was almost non-toxic towards the cells. Onl y at 95 % 0, was a slight damaging effect, of 25 %, observed. The nitr ic oxide ((NO)-N-.) concentrations released from 1 and 2 mM spermineNO NOate were determined as varying between 5 and 12 mu M and between 12 and 22 mu M respectively as measured by the oxyhaemoglobin and the NO cheletropic spin-trapping methods. The decomposition rate of spermineN ONOate and the resulting (NO)-N-. concentrations were independent of O -2 at all applied concentrations. Likewise, the steady-state concentra tions of H2O2 remained at approx. 1 nM at all O-2 concentrations, as m easured with the fluorescent dye scopoletin. L-Tyrosine and L-ascorbat e, both of which are known to scavenge reactive nitrogen species, mark edly diminished spermineNONOate-induced cytotoxicity at 95 % O-2. The formation of 3-nitrotyrosine, indicating the reaction of L-tyrosine wi th nitrogen dioxide ((NO2)-N-.) and/or peroxynitrite anions, was enhan ced in incubations with spermineNONOate at 21 % and 95 % O-2. The resu lts demonstrate that at O-2 partial pressures typically found under ph ysiological conditions and at (NO)-N-. concentrations that can occur i n vivo, (NO)-N-. alone is almost non-toxic towards cultured rat liver endothelial cells. (NO)-N-. at these concentrations in vivo, however, exerts toxic effects at supraphysiological O-2 partial pressures, owin g to its oxidation to reactive nitrogen species such as (NO2)-N-..